Comparative in vitro microdosimetric study of murine- and human-derived cancer cells exposed to alpha particles.

Diffusing alpha-emitter radiation therapy (DaRT) is a proposed new form of brachytherapy using α particles to treat solid tumors. The method relies on implantable ²²4Ra-loaded sources that continually release short-lived α-particle-emitting atoms that spread inside the tumor over a few millimeters. This treatment was demonstrated to have a significant effect on tumor growth in murine and human-derived models, but the degree of tumor response varied across cell lines. Tumor response was found to correlate with the degree of radionuclide spread inside the tumor. In this work we examined the radiosensitivity of individual cells to determine its relationship to tumor response. Cells were irradiated in vitro by α particles using a ²²8Th irradiator, with the mean lethal dose, D0, estimated from survival curves generated by standard methods. The results were further analyzed by microdosimetric tools to calculate z0, the specific energy resulting in a survival probability of 1/e for a single cell, which is considered to better represent the intrinsic radiosensitivity of individual cells. The results of the study demonstrate that, as a rule, tumors that respond more favorably to the DaRT treatment are also characterized by higher intrinsic cellular radiosensitivities, with D0 ranging from 0.7 Gy to 1.5 Gy for the extreme cases and z0 following the same trend.

Secretion of the amyloid precursor protein is elevated isoform specifically by apolipoprotein E4.

Genetic studies suggest that the neuropathology and etiology of Alzheimer's disease (AD) are associated with several genotypes including mutations in the amyloid precursor protein (APP) gene and the allele E4 of apolipoprotein E (apoE). The present study investigated the possibility that cross talk interactions exist between APP and apoE and the extent to which they are affected by the apoE genotype. This was pursued by cell culture and immunoblot experiments utilizing neuroblastoma N2a cells in which the effects of distinct apoE isoforms on the levels of intracellular APP and of secreted APPs were determined. This revealed that treatment of the cells with apoE4, the AD risk factor, resulted in a marked increase in the levels of secreted APPs. This effect was dose dependent (ED50 approximately/= 2.5 microg/ml) and isoform specific in that apoE3 had virtually no effect on the secretion of APPs.

Monoclonal antibodies recognize antigen expressed by osteoblasts.

A marrow stromal osteogenic cell line (MBA-15) was used to create monoclonal antibodies (MoAbs). In this study, we describe a series of MoAbs for mouse marrow stroma (MMS) (MMS-25/17, MMS-85/12, MMS-302/40, and MMS-319/4) that recognized antigens expressed by stromal cells including osteoblastic cells. The MoAbs were screened against various cell and tissue types. MMS-85/12 was positive in detecting an antigen that was highly abundant in osteoblastic cells and primary adherent bone marrow cultures (BMC) but was negative for the marrow adipocytes copartner. The MMS-85/12 MoAb is an IgGl immunoglobulin. The immunohistochemical staining pattern is suggestive of the antigen being associated with the osteoblasts' plasma membrane and with the extracellular matrix constituent secreted by these cells. Western blotting and immunoprecipitation indicated that the antigen that was recognized by MMS-85/12 apparently had a molecular weight of 84 dD.

Central auditory skills in blind and sighted subjects.

Three different central auditory skills were compared and evaluated in 56 blind and 40 sighted subjects. The study consisted of three experiments conducted in three subgroups. Experiment A was performed in order to evaluate the localization function; experiment B for the temporal auditory resolution ability of the blind adult, and experiment C to test the ability of the blind person to discriminate speech material in noise. In all three experiments the blind subjects obtained significantly better results than the sighted subjects. From these results it was concluded that there is supporting evidence of a certain superiority of the blind individual with regard to central auditory function.

Comparison of NK activity in mouse spleen and peripheral blood lymphocytes.

Natural killer (NK) cells originating in mouse peripheral blood were studied with regard to their lytic activity against YAC-1 target cells and to their expression of asialo-GM1 marker on their surface. In Balb/c, CBA/LAK and A/J mice, PBL were found to be approximately twice as effective as splenocytes. Splenic and peripheral NK cells were shown by flow cytometry to have similar lytic potential per cell; the difference in NK activity found in the spleen and in PBL was solely due to the differences in the size of the NK cell population found in the two sites. Strain distribution of NK activity in PBL followed the same pattern observed in splenocytes. The difference in NK activity between CBA and Balb/c mice was shown to be due to the fact that the lytic potential per NK cell was approximately twice as high in the former.

Marker and function analysis of natural killer and alloreactive T cells during early stages of dimethylbenzanthracene carcinogenesis. The immunity system during the precancer period.

The effect of the chemical carcinogen dimethylbenzanthracene (DMBA) on cellular immunity was studied at a 6-mg dose which induces adenocarcinomas and adenoacanthomas in more than 70% of BalB/c mice within 1 year after administration. DMBA caused a significant reduction of splenic natural killer (NK) activity and responsiveness to alloantigens in mixed lymphocyte reactions (MLR). These activities decreased soon after the carcinogen treatment and remained suppressed during the entire tumor induction period. There was a linear correlation between the reduction in NK activity and a selective decrease in the number of asialo GM1 positive cells in the spleen. However, cell sorting experiments using the flow cytometer have shown that the lytic activity per cell of asialo GM-1 positive cells in untreated mice and in DMBA-treated ones was similar. There was no correlation between the suppressed response of the T cells in MLR and the percentage of T cell subpopulations residing in the spleen of the DMBA-treated mice. The decrease in the number of NK cells and the reduced MLR activity in the spleen occurred simultaneously with a decrease in the potential of bone marrow precursor cells to reconstitute NK and MLR activity in the spleen of lethally irradiated mice. These results indicate that the carcinogen DMBA effects the immune system at various levels and either eliminates or inactivates precursor cells as well as mature lymphoid cells.

The analysis of measurements of murine natural killer cell activity.

Several methods of analysing murine NK response measurements have been compared in order to select a quantitative objective measure of NK activity . The fitting of data from 51chromium release experiments to the formula y=A(1-e-kx)((termed the "k method" and shown by Pross et al. (J. Clin. Immunol. (1981) 1,51) to be beneficial in analysing human the NK response) has been particularly evaluated. Computer simulated curves as well as experimental NK dose response curves were analysed testing data in which a plateau level of chromium release had not been reached. Results obtained by the "k method" were very dependent on both the portion of the curve used for the analysis and on small deviations of the data from the theoretical form of the equation. In the analysis of murine NK response the "k method" has no clear advantage over other methods.

B16 melanoma development, NK activity cytostasis and natural antibodies in 3 and 12 month old mice.

Three types of natural immune responses against malignant cells were studied in vitro: Cytotoxicity mediated by splenic NK cells; cytostasis mediated by splenocytes and binding of naturally occurring antibodies to various tumour targets. These responses were studied in untreated 3 and 12 month old mice and in mice of both age groups inoculated with B16 melanoma cells. The results showed that in normal mice NK activity decreases with age, cytostatic activity remains unchanged and the titre of natural antibodies increases. Twelve-month old mice were shown to be appreciably more resistant than 3 month old mice to the development of tumours from subthreshold numbers of B16 tumour cells. In mice injected with threshold amounts of the B16 tumour, there was no change in any of the responses in the tumour-free period, but there was a decrease in NK activity and an increase in cytostatic activity when a large tumour mass developed. An increase in the titre of natural antibodies in young mice injected with the tumour was also seen. The correlation between these changes and tumour appearance and development is discussed.

Natural host defence during oncogenesis. NK activity and dimethylbenzanthracene carcinogenesis.

In this study we determined the effect of administering the chemical carcinogen dimethylbenzanthracene (DMBA) on NK activity mediated by murine splenocytes. The intragastric administration of six weekly 1-mg doses of DMBA to 2- to 3-month-old mice either following a pituitary graft or not, resulted in a tumor incidence which was higher than 60%. The cellularity of the spleens decreased significantly after the third DMBA feeding and the NK activity of these spleens was selectively suppressed. There was no spontaneous reconstitution of the NK activity following cessation of DMBA treatment and during the entire tumor-free period. The adoptive transfer of normal bone marrow, thymus or spleen cells did not restore NK activity in DMBA-treated mice. The NK activity of DMBA-fed mice could be augmented by interferon and by interferon inducers such as poly I:C and dsRNA from Ustilago virus. The effect of these biological response modifiers on NK activity was short-lived but it was possible to restimulate this activity every 24 h. Consecutive treatments of DMBA-fed mice with poly I:C maintained a high level of NK activity for at least 3 weeks.

Some characteristics of natural cytostatic mouse splenocytes.

Murine B16 melanoma cells and metastasis variants of this tumor are resistant to NK activity mediated by normal splenocytes. The B16 cells are, however, sensitive to splenocyte-mediated cytostasis. Cytostasis was measured by a [125I]UDR incorporation-inhibition [(125I]UDR I-I) assay. The main characteristics of the [125I[UDR I-I assay and of the cells mediating it are as follows: The activity is mediated by splenocytes but not by thymocytes, it is not syngeneically restricted and it does not decrease with age. The presence of effector cells is required as splenocyte supernatants or supernatants of effector-target cell mixtures do not cause [125I]UDR I-I. The activity is probably mediated by at least 2 populations of non-phagocytic splenocytes. The first population adheres to plastic surfaces and to Sephadex G-10 columns while the other does not. The sensitivity to [125I]UDR I-I of the high metastasis B16 variant was similar to that of the low metastasis variant.

Natural cellular reactivities mediated by splenocytes from mice bearing three types of primary tumor.

The ability of splenocytes from mice bearing three types of primary tumor to lyse YAC-I target cells (NK activity) and to inhibit [125I]dUrd incorporation ([125I]dUrd I-I = cytostasis) into B16-F10 target cells was compared to the ability of normal splenocytes to perform such activities. The tumor systems used were urethane-induced lung adenomas in BALB/c mice, dimethylbenzanthracene (DMBA)-induced tumors in hormonally-stimulated BALB/c mice and mammary tumors in force-bred C3HeB mice. The two types of natural cellular reactivity in lung adenoma-bearing mice were unaffected. The NK activity of mice bearing DMBA and forced-breeding-induced tumors was suppressed. The cytostatic ability of splenocytes from mice bearing DMBA-induced tumors was significantly elevated. The spleens of mice bearing primary DMBA-induced tumors contained cells able to suppress NK activity or to compete against target cells for NK cells.